Endocrine secretions of pancreatic islets are regulated by complex control mechanisms driven not only by blood-borne metabolites such as glucose, amino acids, and catecholamines, but also by local paracrine influences. The major pancreatic islet hormones, glucagon, insulin and somatostatin, interact with specific pancreatic cell types (A, B, and D cells, respectively) to modulate the secretory response. Although insulin secretion is predominantly controlled by blood glucose levels, somatostatin inhibits glucose-mediated insulin secretion. In addition to inter-islet paracrine regulation of insulin secretion, there is evidence to support the existence of insulinotropic factors in the intestine. This concept originates from observations that glucose taken orally is a much more potent stimulant of insulin secretion than is a comparable amount of glucose given intravenously.
The human hormone glucagon is a 29-amino acid hormone produced in pancreatic A-cells. The hormone belongs to a multigene family of structurally related peptides that include secretin, gastric inhibitory peptide, vasoactive intestinal peptide and glicentin. These peptides variously regulate carbohydrate metabolism, gastrointestinal mobility and secretory processing. However, the principal recognized actions of pancreatic glucagon are to promote hepatic glycogenolysis and glyconeogenesis, resulting in an elevation of blood sugar levels. In this regard, the actions of glucagon are counter regulatory to those of insulin and may contribute to the hyperglycemia that accompanies Diabetes mellitus (Lund, P. K., et al., Proc. Natl. Acad. Sci. U.S.A., 79:345-349 (1982)).
When glucagon binds to its receptor on insulin producing cells, cAMP production increases which in turn stimulates insulin expression (Korman, L. Y., et al., Diabetes, 34:717-722 (1985)). Moreover, high levels of insulin down-regulate glucagon synthesis by a feedback inhibition mechanism (Ganong, W. F., Review of Medical Physiology, Lange Publications, Los Altos, Calif., p. 273 (1979)). Thus, the expression of glucagon is carefully regulated by insulin, and ultimately by serum glucose levels.
Preproglucagon, the precursor form of glucagon, is encoded by a 360 base pair gene and is processed to form proglucagon (Lund, et al., Proc. Natl. Acad. Sci. U.S.A. 79:345-349 (1982)). Patzelt, et al. (Nature, 282:260-266 (1979)) demonstrated that proglucagon is further processed into glucagon and a second peptide. Later experiments demonstrated that proglucagon is cleaved carboxyl to Lys-Arg or Arg-Arg residues (Lund, P. K., et al., Lopez L. C., et al., Proc. Natl. Acad. Sci. U.S.A., 80:5485-5489 (1983), and Bell, G. I., et al., Nature 302:716-718 (1983)). Bell, G. I., et al., also discovered that proglucagon contained three discrete and highly homologous peptide regions which were designated glucagon, glucagon-like peptide 1 (GLP-1), and glucagon-like peptide 2 (GLP-2). Lopez, et al., demonstrated that GLP-1 was a 37 amino acid peptide and that GLP-2 was a 34 amino acid peptide. Analogous studies on the structure of rat preproglucagon revealed a similar pattern of proteolytic cleavage at Lys-Arg or Arg-Arg residues, resulting in the formation of glucagon, GLP-1, and GLP-2 (Heinrich, G., et al., Endocrinol., 115:2176-2181 (1984)). Finally, human, rat, bovine, and hamster sequences of GLP-1 have been found to be identical (Ghiglione, M., et al., Diabetologia, 27:599-600 (1984)).
The conclusion reached by Lopez, et al., regarding the size of GLP-1 was confirmed by studying the molecular forms of GLP-1 found in the human pancreas (Uttenthal, L. O., et al. J. Clin. Endocrinol. Metabol., 61:472-479 (1985)). Their research showed that GLP-1 and GLP-2 are present in the pancreas as 37 and 34 amino acid peptides respectively.
The similarity between GLP-1 and glucagon suggested to early investigators that GLP-1 might have biological activity. Although some investigators found that GLP-1 could induce rat brain cells to synthesize cAMP (Hoosein, N. M., et al., Febs Lett. 178:83-86 (1984)), other investigators failed to identify any physiological role for GLP-1 (Lopez, L. C., et al. supra). The failure to identify any physiological role for GLP-1 caused some investigators to question whether GLP-1 was in fact a hormone and whether the relatedness between glucagon and GLP-1 might be artifactual.
It has now been shown that biologically processed forms of GLP-1 have insulinotropic properties and may delay gastric emptying. GLP-1(7-34) and GLP-1(7-35) are disclosed in U.S. Pat. No. 5,118,666, herein incorporated by reference. GLP-1(7-37) is disclosed in U.S. Pat. No: 5,120,712, herein incorporated by reference.
Variants and analogs of GLP-1 are known in the art. These variants and analogs include, for example, GLP-1(7-36), Gln.sup.9 -GLP-1(7-37), D-Gln.sup.9 -GLP-1(7-37), acetyl-Lys.sup.9 -GLP-1(7-37), Thr.sup.16 -Lys.sup.18 -GLP-1(7-37), and Lys.sup.18 -GLP-1(7-37). Derivatives of GLP-1 include, for example, acid addition salts, carboxylate salts, lower alkyl esters, and amides (see, e.g., WO91/11457). Generally, the various disclosed forms of GLP-1 are known to stimulate insulin secretion (insulinotropic action) and cAMP formation (see, e.g., Mojsov, S., Int. J. Peptide Protein Research, 40:333-343 (1992)).
More importantly, numerous investigators have demonstrated a predictable relationship between various in vitro laboratory experiments and mammalian, especially human, insulinotropic responses to exogenous administration of GLP-1, GLP-1(7-36) amide, and GLP-1(7-37) acid (see, e.g., Nauck, M. A., et al., Diabetologia, 36:741-744 (1993); Gutniak, M., et al., New England J. of Medicine, 326(20):1316-1322 (1992); Nauck, M. A., et al., J. Clin. Invest., 91:301-307 (1993); and Thorens, B., et al., Diabetes, 42:1219-1225 (1993)).
The fundamental defects responsible for causing hyperglycemia in mature onset diabetes include impaired secretion of endogenous insulin and resistance to the effects of insulin by muscle and liver tissue (Galloway, J. S., Diabetes Care, 13:1209-1239, (1990)). The latter defect results in excess glucose production in the liver. Thus, whereas a normal individual releases glucose at the rate of approximately 2 mg/kg/minute, a patient with mature onset diabetes releases glucose at a rate exceeding 2.5 mg/kg/minute, resulting in a net excess of at least 70 grams of glucose per 24 hours.
Because there exists exceedingly high correlations between hepatic glucose production, fasting blood glucose levels, and overall metabolic control as indicated by glycohemoglobin measurements (Galloway, J. A., supra; and Galloway, J. A., et al., Clin. Therap., 12:460-472 (1990)), it is readily apparent that control of fasting blood glucose is essential for achieving overall normalization of metabolism sufficient to prevent hyperglycemic complications. Since existing insulin therapies rarely normalize hepatic glucose production without producing significant hyperinsulinemia and hypoglycemia (Galloway, J. A., and Galloway, J. A., et al., supra) alternative approaches are needed. Thereapy based on administration of GLP-1 analogs is one such approach and is an object of the present invention.
Presently, therapy involving the use of GLP-1 type molecules has presented a significant problem because the serum half-life of such peptides is quite short. For example, GLP-1(7-37) has a serum half-life of only 3 to 5 minutes. Presently, the activity of dipeptidyl-peptidase IV (DPP IV) is believed to readily inactivate GLP-1(7-37) in addition to rapid absorption and clearance following parenteral administration. Thus, there exists a critical need for biologically active GLP-1(7-37) analogs that possess extended pharmacodynamic profiles following parenteral administration.
Accordingly, the primary object of this invention is to provide novel, chemically modified peptides that not only stimulate insulin secretion in type II diabetics but also produce other beneficial insulinotropic responses. The compounds of the present invention persist in the serum for longer periods than native GLP-1(7-37) either by showing resistance to DPP IV or by being absorbed and cleared slower than native GLP-1(7-37) following parenteral administration. Most surprisingly, some compounds of the present invention demonstrated a synergistic effect as individual alterations to GLP-1(7-37) failed to add-up to the biological performance of compounds that contained all of the alterations.